• Gene expression profiles of T cells from hepatitis E virus infected patients in acute and resolving phase.

      TrehanPati, Nirupma; Sukriti, Sukriti; Geffers, Robert; Hissar, Syed; Riese, Peggy; Toepfer, Tanja; Guzman, Carlos A; Sarin, Shiv Kumar; Institute of Liver and Biliary Sciences, D-1, Vasant Kunj, 110 070, New Delhi, India. trehanpati@gmail.com (2011-06)
      Approximately 50% of acute viral hepatitis in young adults and in pregnant women is due to hepatitis E virus (HEV) infection in developing countries. T cell-mediated immune injury probably plays a key role in the pathogenesis of acute hepatitis illness. However, there is a paucity of data on the global gene expression programs activated on T cells, which are subsequently responsible for T cell recruitment to the liver and triggering of immune injury.
    • High-resolution in situ genotyping of Legionella pneumophila populations in drinking water by multiple-locus variable-number tandem-repeat analysis using environmental DNA.

      Kahlisch, Leila; Henne, Karsten; Draheim, Josefin; Brettar, Ingrid; Höfle, Manfred G; Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research (HZI), 38124 Braunschweig, Germany. (2010-09)
      Central to the understanding of infections by the waterborne pathogen Legionella pneumophila is its detection at the clonal level. Currently, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) of L. pneumophila isolates can be used as a tool for high-resolution genotyping. Since L. pneumophila is difficult to isolate, the isolation of outbreak strains often fails due to a viable but nonculturable (VBNC) state of the respective environmental population. Therefore, we developed a cultivation-independent approach to detect single clones in drinking water. This approach is based on the extraction of DNA from drinking water followed by PCR using a set of eight VNTR primer pairs necessary for MLVA genotyping of L. pneumophila. The PCR amplicons were analyzed by single-strand conformation polymorphism (SSCP) and capillary electrophoresis to obtain the respective MLVA profiles. Parallel to the high-resolution analysis, we used the same environmental DNA to quantify the number of L. pneumophila cells in drinking water using real-time PCR with 16S rRNA gene-targeted primers. We used a set of drinking water samples from a small-scale drinking water network to test our approach. With these samples we demonstrated that the developed approach was directly applicable to DNA obtained from drinking water. We were able to detect more L. pneumophila MLVA genotypes in drinking water than we could detect by isolation. Our approach could be a valuable tool to identify outbreak strains even after the outbreak has occurred and has the potential to be applied directly to clinical material.
    • Quantitative reverse transcription polymerase chain reaction analysis of Vibrio cholerae cells entering the viable but non-culturable state and starvation in response to cold shock.

      González-Escalona, Narjol; Fey, Axel; Höfle, Manfred G; Espejo, Romilio T; A Guzmán, Carlos; Vaccine Research Group, Division of Microbiology, GBF-German Research Centre for Biotechnology, Braunschweig, Germany. (2006-04)
      We performed a comparative analysis of the Vibrio cholerae strain El Tor 3083 entering the viable but non-culturable (VBNC) state and starvation after incubation in artificial seawater (ASW) at 4 and 15 degrees C respectively. To this end, we determined bacterial culturability and membrane integrity, as well as the cellular levels of 16S rRNA and mRNA for the tuf, rpoS and relA genes, which were assessed by real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR). Bacterial cells entering the VBNC state showed a 154, 5.1 x 10(3), 24- and 23-fold reduction in the number of copies of 16S rRNA and mRNA for tuf, rpoS and relA, in comparison to exponentially growing cells. The differences were less striking between cells in the VBNC and starvation states. The mRNA for relA was selectively increased in VBNC cells (3.2-folds), whereas a 3.9-fold reduction was observed for 16S rRNA. The obtained results confirmed that key activities of the cellular metabolism (i.e. tuf representing protein synthesis, and relA or rpoS stress response) were still detected in bacteria entering the VBNC state and starvation. These data suggest that the new Q-RT-PCR methodology, based on the selected RNA targets, could be successfully exploited for the identification (rRNA) of V. cholerae and assessment of its metabolic activity (tuf, rpoS, relA mRNA) in environmental samples.